Research Projects

Simultaneous live-cell imaging of chromosome conformation, enhancer-promoter interactions and transcription in mouse ES cells and differentiated derivatives

FMI – Friedrich Miescher Institute for Biomedical Research

Basel

Switzerland

6/13

Supervision

Luca Giorgetti
FMI, 1st Supervisor

Giacomo Cavalli
CNRS, 2nd Supervisor

Objectives

Detect enhancer-promoter interactions and relate them to the dynamics of the chromatin fibre in live mammalian cells — Simultaneously detect transcription from the promoter and understand if, and to what extent, temporal variability in RNA production is the result of stochastic interactions with enhancers.

Methodology

Generation of cell lines that allow the direct visualization of enhancer and promoter DNA using the targeted insertion of arrays of ectopic transcription factor binding sites (e.g. Tet operators or Gal4-UAS sites), bound by the corresponding DNA-binding proteins fused to green and red fluorescent proteins. Simultaneous visualization of several locations along the chromatin fiber between the enhancer and the promoter, using operator arrays inserted at regularly spaced locations between the enhancer and the promoter. Visualization of transcription from the promoter in living cells using MS2 stem-loop repeats. Cell lines will be characterized using high-resolution Hi-C. Live-cell imaging will be performed using wide-field and live STED microscopy.

Expected Results

Enhancers are thought to act on the genes that they control by physically looping on their promoter regions. However, it is unclear how dynamic these interactions are, and whether their dynamics is related to the RNA stochastic production from the promoter, which is commonly observed at most mammalian genes. This question will be addressed by visualising the spatial position of an enhancer and its target promoter in the nucleus, and the conformation of the chromatin fiber in the intervening region, together with RNA transcription from the promoter in live single mammalian cells. Quantitative data analysis and biophysical modelling will allow understanding if and to what extent enhancer-promoter contacts are causal to RNA production.

Planned Secondments

CNRS, France (3-6 months):
Perform live-cell STED microscopy and Hi-C.
CRG (P1b), Marc A. Marti-Renom (3 months):
Statistical analysis of datasets.
BYFACILITY, Spain (2 weeks):
Learning how to communicate complex information.

Enrolment in doctoral programs

Enrolment in FMI International PhD Program.

References

Giorgetti, L. et al. Structural organization of the inactive X chromosome in the mouse. Nature 535, 575-579, doi:10.1038/nature18589 (2016).

Giorgetti, L. et al. Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription. Cell 157, 950-963, doi:10.1016/j.cell.2014.03.025 (2014).

Zhan, Y. et al. Reciprocal insulation analysis of Hi-C data shows that TADs represent a functionally but not structurally privileged scale in the hierarchical folding of chromosomes. Genome Res 27, 479-490, doi:10.1101/gr.212803.116 (2017).